Gross chromosomal rearrangements (GCRs) including chromosome translocations, deletions, inversions, and amplifications are hallmarks and proven sources of many tumors and hereditary diseases in humans. Identification of exogenous and endogenous factors that induce GCRs and underlying mechanisms of chromosome instability are important for our understanding disease prognosis and strategizing approaches for disease prevention and treatment. There are few assays that monitor chromosome instability in human cells and to our knowledge none of them can address complex rearrangements frequently detected in cancers. Moreover, GCRs are often studied in cancer cell lines that have dysregulated cellular metabolism and altered karyotypes, which greatly complicates the analysis of mechanisms of genome instability. The objectives of the proposed research are i) to develop a sensitive assay for detecting different types of chromosomal instability in non-cancerous human cells and ii) to characterize the effect of endogenous and exogenous damaging agents on the induction of GCRs. Specific Aim 1 is to introduce an engineered GCR cassette into two primary immortalized non-cancerous and karyotypically stable male cell lines. We will modify subtelomeric regions of chromosome X in BJ-hTERT fibroblasts and RPTEC/TERT1 renal epithelial cells using a CRISPR/Cas9 system. The GCR assay is based on the use of fluorescent markers well as gene dosage folA marker. This system allows to monitor chromosome X deletions and gene amplification as well as associated rearrangements. Specific Aim 2 is reveal the effect of DNA fragile motifs on the induction of GCRs. The system provides the opportunity to target any sequence motif along with the GCR cassette to the same location on chromosome X. Therefore, any DNA sequence that is suspected of causing double-strand breaks (DSBs) and triggering genome instability can be assessed for its potential to induce deletions and gene amplification in the chromosomal context. We will determine the effect of palindromic sequences and GAA/TTC tracts, which were shown to induce DSBs and chromosomal rearrangements in yeast, on the induction of GCRs in human cells. The resulting genome rearrangements will be analyzed using chromosome spreads coupled with fluorescent in situ hybridization and comparative genome hybridization. siRNA- or CRISPR/Cas9-mediated knockdown will be carried out for genes that have been shown to affect the repeat metabolism or repair processes. Specific Aim 3 is to study the effect of exogenous damage on the induction of GCRs. Cell lines that are under development can be used to determine the effect of any exogenous factors on their ability to trigger GCRs. We will evaluate the frequency of GCRs upon treatment of cells with established DNA-damaging agents such as ionizing radiation, UV-light, methyl methanesulfonate and etoposide. Overall, this unique experimental system will be instrumental in dissecting mechanisms of chromosomal instability in human cells.